Resistin as a new player in the regulation of porcine corpus luteum luteolysis: in vitro effect on proliferation/viability, apoptosis and autophagy.

The formation and luteolysis of the corpus luteum (CL) is strictly controlled by many factors. Imbalance between proliferation and apoptosis processes leads to deficiency of the luteal phase and infertility. Our previous study showed resistin expression in porcine luteal cells and an inhibitory effect on progesterone synthesis. Thus, the aim of the present study was to examine the in vitro effect of resistin on the proliferation/viability, apoptosis and autophagy of porcine luteal cells as well as the involvement of mitogen-activated kinase (MAP3/1), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) in these processes. Porcine luteal cells were incubated with resistin (0.1-10 ng/mL) for 24-72 h and viability was assessed using the alamarBlue or 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the time-dependent effect of resistin on mRNA and protein expression of proliferating cell nuclear antigen (PCNA), caspase 3, BCL2-like protein 4 (BAX), B-cell lymphoma 2 (BCL2), beclin1, microtubule-associated protein 1A/1B-light chain 3 (LC3) and lysosomal-associated membrane protein 1 (LAMP1) was measured by real-time polymerase chain reaction (PCR) and immunoblotting, respectively. We found that resistin enhanced luteal cell viability with no effect on caspase 3 mRNA and protein, increased the BAX/BCL2 mRNA and protein ratio and significantly stimulated the initiation of autophagy, which promotes the maintenance of CL function rather than its regression. Additionally, using pharmacological inhibitors of MAP3/1 (PD98059), AKT (LY294002) and STAT3 (AG490), we observed that the effect of resistin was reversed to the control level in viability and, by influence, MAP3/1 and STAT3 in autophagy. Taken together, our results suggest that resistin, in addition to its well-known effect on granulosa cell function has direct influence on CL luteolysis and the formation and maintenance of luteal cell function.

Cadmium mobility in a circular food-to-waste-to-food system and the use of a cultivated mushroom (Agaricus subrufescens) as a remediation agent.

Cadmium (Cd) and its mobility and toxicity in the food chain receives considerable public and scientific attention. Levels in soil have been increasing, partly caused by mineral fertiliser use. In many areas in the EU, intake of Cd is not far from the maximum tolerable level. Now, urban and more circular approaches to food production are being promoted, in which waste materials substitute growing media and fertilizers. Can this make the situation worse, or is it, on the contrary, possible to extract Cd from the cycle using practical microbial methods? Cd concentration, availability and accumulation in materials and crops in a closed greenhouse system was investigated first. Secondly, we tested if fungal remediation was able to remove a significant amount of Cd. A sequential extraction analysis showed how anaerobic digestion and composting changed chemical Cd availability. Cd levels in the system were comparable to agricultural soils. The vegetable crops did not accumulate Cd. However, the culture of the edible mushroom Agaricus subrufescens in the greenhouse had a strong effect on Cd mobility: It extracted 80% of the Cd from the growing medium into the mushrooms, mostly at the start of fruiting. These early Cd enriched mushrooms can be treated as special waste, while the later harvest complied with EU Cd limits for foodstuffs and can potentially be consumed. Thus, growing this mushroom species in an integrated waste-to-crop system can be a cost-efficient way to remediate Cd from the food chain.

Increased formation of autophagosomes in ectromelia virus-infected primary culture of murine bone marrow-derived macrophages.

Induction of autophagy by ectromelia virus (ECTV) in primary cultures of bone marrow-derived macrophages (BMDMs) was investigated. The results showed that ECTV infection of BMDMs resulted in increased formation of autophagosomes, increased level of LC3-II protein present in aggregates and extensive cytoplasmic vacuolization. These data indicate an increased autophagic activity in BMDMs during ECTV infection.

Adipohormones as prognostric markers in patients with nonalcoholic steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) represents one of the most common liver diseases. It is strongly associated with obesity and insulin resistance and is thought to be part of the metabolic syndrome. NASH can progress to cirrhosis and liver failure. Adipohormones, synthesized in adipose tissue, are involved in the pathophysiology of many acute and chronic liver diseases. The aim of this study was to evaluate the plasma concentrations of adiponectin, resistin, leptin, TNF-alpha and Il-6 in patients with NASH, as well as their correlation with the pathologic parameters. Serum concentration of leptin, adiponectin, resistin, insulin, TNF-alpha, IL-6 were measured with ELISA method. Liver biopsies were obtained from 18 (age 42.55+/-21 years) patients. NASH has been classified according to Dixon score. The control group was represented by 16 non-obese subjects. Mean serum concentration of adiponectin in patients with NASH was significantly lower than in healthy subjects (4.87+/-1.96 vs. 8.33+/-4.56 ng/ml; p<0.05). Mean serum levels of TNF-alpha in patients with NASH were significantly higher than in controls (34.2+/-19.7 vs. 20.7+/-15.5 ng/ml; p<0.05). In patients with more advanced inflammation (grade 2-3) and fibrosis (stage 2) in pathology, serum concentration of leptin was significantly higher than in patients with steatosis and less advanced inflammation (grade 1) and fibrosis (stage 1) (median 8.94 vs. 16.2 ng/ml; p<0.05). No significant differences of serum concentration of others adipohormones between these two groups of patients were stated. Moreover, we observed the correlation in serum levels (examined group vs controls) between: resistin and TNF-alpha (r = 0.62; p<0.05), adiponectin and IL-6 (r = -0.60; p<0.05) and leptin and insulin (r = -0.51; p<0.05). In conclusion, based on our study we speculate that changes of adipohormones levels may be markers of NASH and the serum level of leptin can be associated with more advanced form of NASH.

Induction of insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells by cells of trophoblast origin.

OBJECTIVE: To determine the influence of the conceptus on the induction of decidualization in endometrial stromal cells from the baboon. METHODS: For in vivo studies, implantation sites from day 22 of pregnancy in the baboon were analyzed for insulin-like growth factor-II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) mRNAs using in situ hybridization. For in vitro studies, Jeg-3 cells or primary cytotrophoblasts isolated from term placenta were cocultured with a monolayer of stromal cells from the baboon endometrium. Cytotrophoblasts were placed either directly on top of the stromal cell monolayer or in transwell cell culture inserts and cultured for 7 days. No exogenous hormones were added. Stromal cells were analyzed for IGFBP-1 mRNA and protein by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. RESULTS: Examination of early implantation sites in the baboon revealed a high expression of IGF-II mRNA in the invading cytotrophoblasts. Conversely, the stromal cells of the endometrium directly adjacent to the cytotrophoblasts expressed IGFBP-1 mRNA. Endometrial stromal cells cocultured with Jeg-3 cells or primary cytotrophoblasts for 7 days expressed IGFBP-1 mRNA and protein. This expression occurred in both the direct coculture system and coculture using the cell culture inserts. Cytotrophoblasts also induced the expression of prolactin in stromal fibroblasts following coculture. CONCLUSIONS: The conceptus is capable of inducing decidualization of endometrial stromal cells. This is shown in both the in vivo and in vitro systems.

Prevalence of BRCA1 founder mutations in western Poland.

The frequency of three BRCA1 founder mutations was examined in a group of Polish breast/ovarian cancer patients and women at increased risk of these cancers based on family history. Among the 15 mutations found (5385-5386insC, 187-188delAG, and 4154delA), eight were detected in women with a low or moderate family history. The frequency of the IVS20+48ins12 variant was also analyzed and its distribution within risk groups argues against its involvement in cancer predisposition.

Allelic imbalance of BRCA1 transcript in the IVS20 12-bp insertion carrier.

One of the unclassified variants of the BRCA1 gene which has drawn considerable attention in recent years is the 12-bp insertion/duplication in intron 20. In this report, we show that a contribution from one chromosome cannot be detected in the BRCA1 transcript of the 12 bp insertion carrier. We also demonstrate here that the single transcript variant we observe by cDNA analysis originates from the same BRCA1 allele that harbours the 12-bp insertion. Hum Mutat 16:371, 2000.

High frequency of recurrent mutations in BRCA1 and BRCA2 genes in Polish families with breast and ovarian cancer.

Germ-line mutations in BRCA1 and BRCA2 genes result in a significantly increased risk of breast and ovarian cancer. Other genes involved in an increased predisposition to breast cancer include the TP53 gene, mutated in Li-Fraumeni syndrome. To estimate the frequency of germ-line mutations in these three genes in Upper Silesia, we have analyzed 47 breast/ovarian cancer families from that region. We found five different disease predisposing mutations in 17 (36%) families. Twelve families (25.5%) carried known BRCA1 mutations (5382insC and C61G), four families (8.5%) carried novel BRCA2 mutations (9631delC and 6886delGAAAA), and one family (2%) harbored novel mutation 1095del8 in the TP53 gene, which is the largest germline deletion in coding sequence of this gene identified thus far. The 5382insC mutation in BRCA1 was found in 11 families and the 9631delC mutation in BRCA2 occurred in three families. These two mutations taken together contribute to 82% of all mutations found in this study, and 30% of the families investigated harbor one of these mutations. The very high frequency of common mutations observed in these families can only be compared to that reported for Ashkenazi Jewish, Icelandic, and Russian high-risk families. This frequency, however, may not be representative for the entire Polish population. The observed distribution of mutations will favor routine pre-screening of predisposed families using a simple and cost-effective test.

Postoperative plasma interleukin-6 in patients with renal cancer correlates with C-reactive protein but not with total fibrinogen or with high molecular weight fibrinogen fraction.

The concentration of fibrinogen (Fb) and its fractions, the levels of interleukin-6 (I1-6), C-reactive protein (CRP), and immunoglobulin G (IgG) were determined in 38 patients operated on because of renal cancer. The increased Fb and I1-6 concentrations were found in approximately one-half of the patients with malignancy. The relations among the high molecular weight (HMW) and two low molecular weight (LMW and LMW') fibrinogen fractions in these patients before surgery did not differ from the corresponding relations in normal subjects. The levels of all (except IgG) compounds studied increased after surgery and the peak of I1-6 was observed on the first postoperative day but that of CRP on the third day. The concentrations of total Fb and of its HMW fraction were the highest also on the third postoperative day and this was in contrast with the decline of low molecular weight fractions at the same time. These variations of estimated variables can be regarded as being relevant to the acute phase response. We have noted a correlation between the maximal concentrations of I1-6 and CRP, but not between the corresponding concentrations of Il-6 and total Fb or HMW Fb; this may suggest that the concentration of Fb is also under the control of a factor other than I1-6.

Novel BRCA1 mutations and more frequent intron-20 alteration found among 236 women from Western Poland.

Three different novel BRCA1 mutations, five independent cases of the same 12 bp insertion-duplication in intron-20 and two novel rare BRCA1 sequence variants were identified among 122 Polish women with positive, in most cases moderate family history of breast and/or ovarian cancer, 80 controls and 34 unselected breast cancer tissue specimens. All mutations and variants were germline. The 4153 delA frameshift mutation, the Tyr105Cys missense mutation and two cases of the alteration in intron-20 were found in the group of healthy women with positive family history. Two other cases of the intronic insertion were found in unselected controls. Their carriers had no family history of breast or ovarian cancer but other cancers occurred in their families. The 1782 Trp/STOP nonsense mutation and one case of the insertion in intron-20 were first found in tissue specimens of breast cancer patient and breast/ovarian cancer patient, respectively. Their carriers also had no family history of breast or ovarian cancer. The distribution of the insertion in intron-20 in analysed groups and results of RT-PCR experiments suggest a less prominent role for this variant considered earlier a splicing mutation. This study shows also, that more population-oriented research is needed, involving women with less profound or even no family history of breast and ovarian cancer, to better understand the role and significance of different BRCA1 variants and mutations.

[Transferrin and ferritin as laboratory indices of body iron deposits]. / Transferyna i ferrytyna jako laboratoryjne wskazniki stanu zasobów zelaza w organizmie.

The aim of the study was to present the relation between serum parameters connected with iron metabolism; serum iron, transferrin and ferritin. Biochemical data in 126 patients were not compared with microscopic evaluation of erythrocytes. The tendency to decrease of serum transferrin with increased of serum ferritin was found. Significant relations between those proteins and serum iron were observed only in patients with very low ferritin values. It can be concluded that ferritin and transferrin may be considered as independent diagnostic in differential diagnosis of real (latent) and false iron depletion.

Occurrence of the low-mobility H1 histones subfraction in embryonic, differentiated, and neoplastic tissues of the Syrian hamster.

Electrophoretically slow H1 histone subfractions with mobilities identical to that of the subfraction found in the Kirkman-Robbins hamster hepatoma chromatin have been shown to be present in 12-day hamster embryos and in a sarcoma-type hamster tumor induced by SV40. No subfractions of such mobility were found in hamster liver, regenerating liver, thymus, spleen, and a fast-growing transplantable amelanotic hamster melanoma. A suggestion is made that some defective mechanisms of differentiation may affect the regulation of expression of the genes coding for the H1 histone subfractions. The same mechanisms may possibly but not necessarily be connected with the molecular events leading to neoplastic growth.